The polymerase chain reaction (PCR) is a widely-used clinical laboratory procedure for sequence-specific target amplification. However, contamination is an ongoing problem. For many PCR applications, it is essential that the only DNA that enters the reaction is the template to be amplified.
Increases in the sensitivity and specificity of PCR have enabled analysis of heterogeneous DNA (e.g., from tumor biopsies, stool). The DNA to be amplified is typically a rare event in the context of a heterogeneous sample. However, as the degree of sample heterogeneity increases, the tolerable threshold of background (signal generated from a negative control sample) becomes increasingly lower. This is necessary to retain a sufficient signal to noise ratio between positive clinical samples and negative control samples within an assay, and therefore to retain high confidence in the assay results. The end result of applying PCR to more heterogeneous DNA environments is a reduced tolerance for pre-PCR contamination from previous amplified material. Currently, there are three methods applied to prevent PCR contamination: (1) physical separation of the sample, pre-PCR setup, and post-PCR manipulations; (2) use of Uracil DNA-glycosylase and dUTP instead of dTTP, and (3) the use of UV irradiation.
Thousands of samples may be analyzed in a single clinical assay with multiple PCR negative controls added. In this context, an investigator relies on the presence or absence of amplified product within a limited number of negative control samples to confirm the origin of amplification products observed in experimental samples. If only one PCR negative control sample is positive, the entire assay is invalid, and must be repeated. In an assay containing 1000 samples, each sample must be run with another set of negative controls when contamination is observed.
However, the mere lack of amplification product within the PCR negative control is not determinative of a positive PCR result in a sample in which contamination is rare. This kind of sporadic contamination is especially problematic in an extremely large throughput assay in which 5 to 10 negative controls are run for approximately every 1000 samples. Statistically, the likelihood of sporadic contamination in, for example, 1000 samples will not be detected in only 5 negative controls. Sporadic contamination is also a significant problem when PCR based analyses are performed on heterogeneous (rare event analysis) samples in which a positive result is generated from, for example, 1-5% of the total amplification product present within the sample. Generally, within a PCR based inherited disease diagnostic assay, given the 50% heterogeneity that exists in any genomic DNA sample, a 1-5% increase in signal in a true negative sample would appear as a slight increase in background, but would not indicate a false positive result. However, within an assay involving samples with heterogeneous populations of DNA, a 1-5% positive signal generated by a true negative sample would result in a false positive.
In addition, even within an inherited disease diagnostic assay, if there were 1000 samples analyzed and 5-10 negative control PCR reactions were run in parallel, and one or two of the negative control samples were positive, results from any of the samples themselves would be compromised. If the contamination of the PCR negative control samples is truly sporadic, then repeat analysis of all 1000 samples is probably not necessary and extremely costly. The lack of amplification product within the PCR negative control samples is not determinative that a positive PCR result within an experimental sample set is not from rare (sporadic) contamination that has occurred in only a few samples within the assay (and not due to the negative controls run in parallel).
In many assays, “normal” PCR contaminants (e.g., resulting from purification problems) are an even greater hindrance and leads to decreased sensitivity of the assay. These “normal” PCR contaminants can lead to false negative results that undermine the accuracy of (and confidence in) the particular assay.
Therefore, methods are needed for performing clinical analyses on samples of DNA heterogeneity (e.g. sporadic cancer detection) such that sporadic contamination from previous amplification product or “normal” PCR contaminants do not result in false positive or false negative results.